Chemical constituents from Carica papaya Linn. leaves as potential cytotoxic, EGFRwt and aromatase (CYP19A) inhibitors; a study supported by molecular docking

The phytochemical investigation of the hydromethanolic extract of Carica papaya Linn. leaves (Caricaceae) resulted in the isolation and characterization of ten compounds, namely; carpaine (1), methyl gallate (2), loliolide (3), rutin (4), clitorin (5), kaempferol-3-O-neohesperidoside (6), isoquercetin (7), nicotiflorin (8) and isorhamnetin-3-O-β-d-glucopyranoside (9). The compounds 2, 3, 5–7 and 9 were isolated for the first time from the genus Carica. An in vitro breast cancer cytotoxicity study was evaluated with an MCF-7 cell line using the MTT assay. Methyl gallate and clitorin demonstrated the most potent cytotoxic activities with an IC50 of 1.11 ± 0.06 and 2.47 ± 0.14 μM, respectively. Moreover, methyl gallate and nicotiflorin exhibited potential EGFRwt kinase inhibition activities with an IC50 of 37.3 ± 1.9 and 41.08 ± 2.1 nM, respectively, compared with the positive control erlotinib (IC50 = 35.94 ± 1.8 nM). On the other hand, clitorin and nicotiflorin displayed the strongest aromatase kinase inhibition activities with an IC50 of 77.41 ± 4.53 and 92.84 ± 5.44 nM, respectively. Clitorin was comparable to the efficacy of the standard drug letrozole (IC50 = 77.72 ± 4.55). Additionally, molecular docking simulations of the isolated compounds to EGFR and human placental aromatase cytochrome P450 (CYP19A1) were evaluated. Methyl gallate linked with the EGFR receptor through hydrogen bonding with a pose score of −4.5287 kcal mol−1 and RMSD value of 1.69 Å. Clitorin showed the strongest interaction with aromatase (CYP19A1) for the breast cancer receptor with a posing score of −14.2074 and RMSD value of 1.56 Å. Compounds (1–3) possessed a good bioavailability score with a 0.55 value.


Introduction
Pawpaw or Papaya (Carica papaya Linn.) is an evergreen tree that belongs to the family Caricaceae, it is indigenous to Central America and the South of Mexico. It is commonly grown in the subtropical and tropical regions and cultivated in many countries worldwide. 1,2 C. papaya is considered the most valuable species in the family due to its nutritional and therapeutic benets. 3 Its leaves are traditionally used as a cardiotonic, vermifuge, febrifuge and as a treatment for colic, dengue fever, beriberi, asthma, cancer and stomach troubles in India and Australia. 4,5 In addition, the different parts of the Pawpaw plant have been used for their therapeutic applications and strong activities as an antibacterial, antiviral, antitumor, antidiabetic, anti-inammatory and management of neurodegeneration. 6 Numerous scientic studies have conrmed that C. papaya contains alkaloids, glycosides, tannins, saponins, avonoids and glycosides which may be responsible for its therapeutic activity. 5 Cancer is a major cause of mortality with a higher incidence in developed and developing countries. Worldwide, about 19.3 million new cancer cases and an estimated 10.0 million deaths due to cancer were reported in 2020. Female breast cancer was the most commonly diagnosed cancer, with an estimated 11.7% of new cases. 7 In Egypt, breast cancer incidence accounts for about 38.85% of total diagnosed female cancer cases. 8 Despite the advances in cancer research and clinical trials of promising new therapies, there is still a great demand for the discovery of new safe and effective drugs with low adverse effects on human health. 9 Natural products have a strong role in the development of anti-cancer agents, thus various drug discovery programs continue to invest in this outstanding source. 10 Many scientic studies have reported the effect of C. papaya leaves extract on the treatment of breast cancer, cervical carcinoma, hepatocellular carcinoma, osteosarcoma, lung adenocarcinoma and many other types of cancer. 6,11 The EGFR tyrosine kinase is critical for hormone receptor positive breast cancer and upregulation of EGFR leads to aberrant signalling. 12 It has been reported that 57% of breast carcinomas express EGFR wt . 13 In addition, triple negative breast cancer (TNBC) characterized by low expression of estrogen, progesterone and Her2 receptors, is associated with overexpression of EGFR. 14 In addition to the role of EGFR in breast cancer progression, aromatase enzyme is critical for breast cancer development and progression. Aromatase catalyzes the nal rate-limiting step in estrogen biosynthesis. Aromatase catalyzes a three-step reaction on androgen substrates. The third step of the reaction leads to the aromatization of the A-ring. Aromatase is also highly expressed in breast cancer producing higher levels of estrogen. 15 However, breast cancer cells constantly develop resistance to aromatase inhibitors by acquiring estrogen receptor mutations, truncation and upregulation of ER-related transcription factors activator protein 1 (AP1) and NF-kB, aromatase inhibitors are effective in breast cancer treatment. [16][17][18] Even though previous studies on the effect of C. papaya and its leaves on breast cancer, the effect of its bioactive compounds and possible mechanism of action on specic cancer targets still needs more exploration. This provoked us to carry out an extensive phytochemical study of C. papaya Linn. leaves to isolate the active metabolites and test their effect on the MCF-7 breast cancer cell line as well as evaluation of their epithelial growth factor receptor (EGFR wt ) kinase and aromatase (CYP19A) enzyme inhibition activity. In addition, the explanation of the potent compounds possible binding mode to their targets by in silico molecular docking studies.

Structure elucidation of the isolated compounds
The chemical structures of compounds (1-9) was determined based on different spectroscopic data including 1D and 2D NMR, MS data, as well as comparison of the data with the previously reported in the literature. The structures of all the isolated compounds are declared in Fig. 1. The compounds were elucidated as carpaine (1)

MTT cytotoxicity assay in breast cancer cells line (MCF-7)
Breast cancer is a common malignancy among females linked with several risk factors as age, familial factors, reproductive factors, lifestyle and hormonal factors. Breast cancer usually starts from ductal hyperproliferation and then develops into either benign or metastatic tumors inuenced by exposure to carcinogens. 28 Extensive studies investigated the role of dietary and natural products in the reduction of development and progression of breast tumors. Natural products exhibit several anticancer activities by different mechanism such as direct inhibition of tumor cell proliferation, metastasis and angiogenesis of breast tumor cells. 29 The cytotoxic effect of the isolated compounds from C. papaya on MCF-7 cells line was evaluated using MTT assay which measures metabolic activity as an indicator of cellular viability and proliferation. The results ( Table 1) showed that methyl gallate (2) and clitorin (5) exhibited the most potent cytotoxic effects against MCF-7 cell lines with the IC 50 value 1.11 AE 0.06 and 2.47 AE 0.14 mM, respectively. Moreover, kaempferol-3-O-neohesperidoside (6), nicotiorin (8) and isorhamnetin-3-O-b-D-glucopyranoside (9) showed strong effect with IC 50 values higher than the standard drug staurosporine (IC 50 ¼ 10.2 AE 0.58) ( Table 1). The possible mechanism of their potential effect should be investigated for the development of targeted drug therapy for breast tumors.

EGFR wt kinase activity
Herein, we studied the inhibitory activity of the isolated compounds on EGFR wt . 30,31 The results ( Table 2) revealed that all the tested compounds possess EGFR wt potent inhibition activity at nM concentration with IC 50 values ranging from 37.3 AE 1.9 to 100.20 AE 5.1 nM. Methyl gallate (2) and nicotiorin (8) exhibited the highest inhibitory activities comparable with erlotinib (IC 50 ¼ 35.94 nM) with IC 50 values of 37.3 AE 1.9 and 41.08 AE 2.1 nM, respectively. On the other hand, compounds 1, 3-7 and 9 had an interference effect with EGFR wt but to lesser extent than methyl gallate (2) and nicotiorin (8).

Aromatase (CYP19A) enzyme activity
Breast cancer is mostly reliant on estrogen or progesterone, especially in postmenopausal females. Generally, there are two common treatment approaches for breast cancer, modulation of estrogen receptor by selective estrogen receptor modulators or inhibition of aromatase enzyme by aromatase inhibitors. Aromatase is the key enzyme that acts on androgen precursors for the synthesis of estrogen. Aromatase inhibitors are used to either block the production of estrogen or block the action of estrogen on its receptors and for treatment of estrogen dependant breast cancer. 32,33 In the current study, the investigation of the effect of isolated compounds on aromatase (CYP19A) revealed that all the tested compounds potently inhibited the effect of aromatase enzyme with IC 50 range from 77.41 AE 4.53 to 436.40 AE 25.6 nM ( Table 2).
The IC 50 values of compounds clitorin (5) and nicotiorin (8) Table 2. It is noteworthy that clitorin (5) was more effective than the standard drug letrozole (IC 50 ¼ 77.72 AE 4.55). In addition, the nicotiorin (8) showed a dual potent effect on both EGFR and aromatase comparable to the standard drugs which could be scaffold to the development of safe effective therapy for breast cancer.

ADME pharmacokinetics and drug-likeness properties
The nine identied compounds were screened for their ADME pharmacokinetics and drug-likeness using the websites servers 36,37 as previously described 38 (Table 3). All tested compounds had good membrane permeability (log p values #5). The compounds (1-3) possessed good bioavailability scores with 0.55 value. Furthermore, compounds (4-9), showed promising drug-likeness scores.
Detailed molecular properties, absorption, distribution, metabolism and excretion in silico assessment are shown in Table 3.

General experimental procedures
1 H-and 13 C-NMR spectra were acquired at 25 C using a Varian Inova 400 MHz NMR spectrometer. High-resolution mass

EGFR wt kinase inhibition activity
Evaluation of the inhibitory activity of the isolated compounds (1-9) against EGFR wt kinase was carried out using EGFR wt Kinase Assay Kit (BPS biosciences) according to manufacturer's instructions. 40,41 In brief, the master mixture was constructed from EGFR wt enzyme, their substrates, ATP and kinase assay enzymatic buffer were incubated with the tested compounds for 40 min at 30 C to achieve the enzymatic reaction. Then, the reaction was stopped by the addition of a detecting reagent (Kinase-Glo Max reagent), followed by incubation at room temperature for another 15 min. Finally, luminescence was measured using the microplate Robonik P2000 ELISA Reader.
All samples and controls were tested in duplicate and the results are presented as percentage enzyme inhibition and compared to erlotinib selected as reference drugs due to their potent inhibitory activity of EGFR wt .

Aromatase (CYP19A) inhibition activity
The in vitro aromatase inhibitory activity of the compounds (1-9) was evaluated using (Bio Vision, Aromatase (CYP19A) Inhibitor Screening Kit (Fluorometric)) in comparison to letrozole as the reference drug. This method was carried out according to Acar Çevik et al., 2020 procedure. 42 The compounds were dissolved in DMSO in concentrations ranging from 10 to 10 4 nM. The recombinant human aromatase stock was prepared according to the protocol and the samples were added and the plate was incubated for 10 min at 37 C to allow tested compounds to interact with the aromatase. Aer incubation, 30 mL of the aromatase substrate/NADP + mixture was added to each well and the uorescence at E x /E m ¼ 488/527 nm was measured immediately (within 1 min).

Molecular docking simulation
Molecular docking was performed as briey described to understand the binding affinity of the potent compounds in comparison with the reference drugs at the molecular level. 43 The crystal structure of both the epidermal growth factor receptor tyrosine kinase (EGFR wt ) (PDB ID: 1M17) and Human placental aromatase cytochrome P450 (CYP19A1) for the breast cancer receptor (PDB ID: 3S79) were obtained from the Protein data bank. The molecular docking simulation was conducted using the "Molecular Operating Environment (MOE 2014.9) and preparation and optimization of both ligands and receptors were carried out according to induced t MOE protocol. 44 The parameters of scoring were Triangle Matcher, scoring was set at London dG and rescoring at GBVI/WSA dG. The docking score, root mean square deviation (RMSD) and ligand-receptor complexes were tested for interaction analysis. The 3D images were made using the MOE visualizing tool.

Drug like properties and ADME prediction of isolated compounds
The drug likeliness, molecular properties prediction ADME and pharmacokinetic parameters of the isolated compounds were calculated using a set of soware including "MolSo," "Molinspiration", "PreADME" and "SwissADME" websites servers. 36,37

Statistical analysis
Results are expressed as mean AE standard deviation (SD) based on triplicate experiments. The IC 50 values of the tested compounds were determined using curve tting in the R programming language and associated packages including Magrittr, 45 drc 46 and ggplot2. 47 Graphs and gures were generated using R console and PyMOL (PyMOL Molecular Graphics System, Schrödinger).

Conclusion
Due to the reported various secondary metabolites of C. papaya Linn. leaves, in addition to their promising cytotoxic activity against breast cancer cell line supported by protein kinase inhibition activities and molecular docking study. They could be considered as potential candidates against breast cancer. Therefore, further investigations could have a supportive role in the pharmaceutical eld towards the development of new breast anti-cancer drugs.